Structural dynamics of oligodendrocyte lysis by perforin in culture: relevance to multiple sclerosis

The mechanism by which oligodendrocytes are depleted from active lesions in multiple sclerosis (MS) is not clear but many reports implicate a cytolytic process. The most applied animal model for MS, chronic relapsing experimental autoimmune encephalomyelitis (EAE), has been established in inbred strains of mice, especially SJL and PL. Studies on oligodendrocytes from these strains in vitro have been hampered to date by an inability to grow these cells from mouse CNS tissue. We report here a successful method to culture SJL mouse oligodendrocytes and have analyzed lysis of these cells in vitro mediated by the pore-forming protein, perforin, a candidate effector molecule in inflammatory demyelination. Cultures were exposed to murine perforin, 36-72 hemolytic U, for up to 2.5 hr and examined using the oligodendrocyte phenotypic markers O4, galactocerebroside and myelin basic protein (MBP), in addition to a membrane dye (DiI) and a marker of necrosis, propidium iodide, (PI). Cultures were imaged chronologically by phase contrast, immunofluorescence, digital, light and electron microscopy. Findings showed that the majority of oligodendrocytes were killed within 60-90 min via pore expansion and ultimately, membrane disruption. The structural features of the cellular damage comprised swelling of the cell body, fenestration and fragmentation of membranes and processes, cytoplasmic vacuolation and breakdown of the nuclear envelope. Astrocytes in the same system were relatively resistant to cell lysis. The above patterns of oligodendrocyte damage in SJL oligodendrocytes were reminiscent of patterns in the MS lesion, leaving us to conclude that perforin may play an important role in the human disease.